Methods of Treating Colorectal Cancer

ABSTRACT

Disclosed herein are methods for treating/and or preventing colorectal cancer using a specific inhibitor of SMAD7 expression or function. Also disclosed are pharmaceutical compositions containing an inhibitor of SMAD7 for treating and/or preventing colorectal cancer and manufacture of medicaments containing an inhibitor of SMAD7 to be used in treating and/or preventing colorectal cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/211,471, filed Mar. 14, 2014, which claims priority to and thebenefit of U.S. Provisional Application No. 61/790,488, filed Mar. 15,2013, the entire contents of each of which are herein incorporated byreference for all purposes.

FIELD OF THE INVENTION

The present invention is generally directed towards methods of treatingand/or preventing colorectal cancer and colorectal cancer cell growthvia administration of SMAD7 inhibitors, particularly antisenseoligonucleotides directed against SMAD7, as well as pharmaceuticalcompositions containing SMAD7 inhibitors for use in treating colorectalcancer.

BACKGROUND

Colorectal cancer is a disease characterized by unchecked proliferationof cells of the large intestine, including cells of the colon or rectum.Colorectal cancer tumors are believed to originate in normal mucosa.Tumorigenesis is associated with the appearance of clusters of enlargedcrypts showing proliferative and biochemical abnormalities.Proliferation of the epithelial cells that carry the causative mutationor mutations can become early stage tumors characterized by high-gradedysplasia. Further growth can result in invasive growth into the musclelayers and through the bowel wall. If untreated, these tumors can spreadto regional lymph nodes and then metastasize to distant sites, at whichpoint they become largely untreatable using currently availabletechnologies (Markowitz and Bertagnolli (2009) N. Engl. J. Med. 361(25):2449-2460). While tumors can arise de novo, evidence indicates thatapproximately 60% of carcinomas originate from pre-existing adenomas(Soreide et al., (2011) Discov. Med. 12(66):393-404). Thus, the vastmajority of colorectal cancer tumors can be classified asadenocarcinomas, but lymphomas and squamous cell carcinomas are alsoobserved in a smaller subset of cases. Genetic mutations that result incarcinogenesis include mutations in members of the Wnt signalingpathway, members of the TGF-β cell signaling pathway such as TGF-β1 andSMAD family members, proteins that regulate the balance between cellproliferation and cell death such as TP53, and other proteins such asDCC (Reya and Clevers (2005) Nature 434(7035):843-850; Baker et al.,(1989) Science 244:217-221; Markowitz and Bertagnolli, supra). AbnormalPI3K/Akt activation and downstream mTOR signaling are associated withcolorectal cancer tumorigenesis (Rychahou et al., (2006) Ann. Surg.243(6):833-842). High levels of EGFR expression have also been observedin colon cancer cell lines and are correlated with colorectal cancertumor progression (Ciardiello et al., (1991) Proc. Natl. Acad. Sci. USA88(17):7792-7796). Beyond familial and genetic factors, risk factors forcolorectal cancer may include low levels of physical activity, alcoholconsumption, high dietary intake of fat and meat and low intake of fiberand vegetables. Symptoms of colorectal cancer typically include rectalbleeding, anemia, constipation, blood in the stool, weight loss, fever,loss of appetite, and nausea or vomiting.

Colorectal cancer is the second most common form of cancer amongAmerican male and female survivors of cancer (Siegel et al., (2012) CACancer J. Clin 62(4):220-41). Additionally, colorectal cancer is amongthe top three most common causes of cancer death in the Western world(Soreide et al., (2011) Discov. Med. 12(66):393-404). The more recentadaptation of many Asian countries to a Western lifestyle has alsoresulted in a significant increase in colorectal cancer in thosepopulations (Yang et al., (2011) Dig. Surg. 28(5-6):379-385). In 2012,it is estimated that there were 1.2 million individuals in the UnitedStates living with a previous diagnosis of colorectal cancer. For thesame year, it was predicted that an additional 143,460 would bediagnosed with the disease. The median age at diagnosis of colorectalcancer is 68 years for males and 72 years for females (Howlader et al.,(2011) SEER Cancer Statistics Review, 1975-2008. Bethesda, Md.: NationalCancer Institute). While incidence of colorectal cancer is not rare inelderly adults, only 59.1% of individuals over the age of 50 receivecolorectal cancer screening according to guidelines (American CancerSociety (2012) Cancer Prevention & Early Detection Facts & Figures.Atlanta, Ga.: American Cancer Society). This lack of early detectionresults in only 39% of patients being diagnosed when the cancer has notprogressed past a local stage (Howlader et al., supra). Given theincreasing number of patients suffering from colorectal cancer, there isa need for development of robust treatment methods, especially for thelarge number of patients who are not identified by early screening.

SUMMARY

The invention described herein provides novel methods for treatingcolorectal cancer via inhibition of SMAD7, leveraging the role of SMAD7as a key antagonist of the TGF-β signaling pathway. While otherpotential targets for therapeutic intervention in colorectal cancer havebeen proposed, the present invention provides a new treatment shown toprevent, retard, stop, or reverse colorectal tumor cell growth.

The present invention provides a method for treating colorectal cancerby inhibiting SMAD7. Specifically, the invention provides a method ofinhibiting SMAD7 in a colorectal tumor in a patient. The invention alsoprovides a method of inhibiting growth of colorectal cancer cells byinhibiting SMAD7. The invention also provides a method for inhibitingSMAD7, treating colorectal cancer, and/or inhibiting growth ofcolorectal cancer cells via administration of an effective amount of aninhibitor of SMAD7. For example, inhibitors of SMAD7 (e.g., anti-SMAD7antisense therapies, i.e., antisense oligonucleotide against SMAD7, andantibodies against SMAD7). “Antisense oligonucleotide,” as used herein,refers to a short synthetic oligonucleotide sequence complementary tothe messenger RNA (mRNA), which encodes for the target protein (e.g.,SMAD7). Antisense oligonucleotide sequences hybridize to the mRNAproducing a double-strand hybrid that can lead to the activation ofubiquitary catalytic enzymes, such as RNase H, which degrades DNA/RNAhybrid strands thus preventing protein translation.

An inhibitor of SMAD7 may be a specific inhibitor of SMAD7 such as anantisense oligonucleotide or any other means of targeting SMAD7 with ahigh degree of specificity. An antisense oligonucleotide inhibitor ofSMAD7 may be selected from, but is not limited to, the group of SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12,described herein. For example, an antisense oligonucleotide inhibitor ofSMAD7 may include SEQ ID NO: 5 or SEQ ID NO: 9, or may include SEQ IDNO: 6 or SEQ ID NO: 10. An exemplary SMAD7 antisense oligonucleotide ofthe present invention is the sequence represented by a form of SEQ IDNO: 6, in which all phosphate bonds are phosphorothioate bonds (SEQ IDNO: 10, referred to herein as GED-0301).

“Inhibitor,” as used herein, refers to an agent capable of decreasingexpression of a gene or DNA sequence, preventing or suppressingproduction, activity, or translation of an RNA product of a gene intoprotein, or preventing or suppressing the activity of the proteinproduct of a gene, through either a direct or indirect interaction withthe gene, RNA product, or protein product of a gene or any transitionalforms of these entities or another molecular entity whose activity orexpression impinges upon the activity or expression of the intendedtarget. Such inhibitors may include, but are not limited to, forexample, antibodies, small molecules that bind to a specific moleculartarget, and antisense oligonucleotides targeted to specific mRNAtranscripts. Accordingly, “inhibitor of SMAD7,” as used herein, refersto an agent capable of decreasing expression of SMAD7, preventing orsuppressing production, activity, or translation of an RNA product ofSMAD7 into protein, or preventing or suppressing the activity of theprotein product of SMAD7, through either a direct or indirectinteraction with the gene, RNA product, or protein product of SMAD7 orany transitional forms of these entities or another molecular entitywhose activity or expression impinges upon the activity or expression ofSMAD7.

The present invention also provides for methods of treating colorectalcancer via administering specific inhibitors of SMAD7. A “specificinhibitor,” as used herein, refers to an agent that has structuraland/or functional properties that allow it to exclusively or with a highdegree of selectivity act upon a molecular target. Thus, a specificinhibitor of SMAD7 possesses the inherent functional property oftargeting the SMAD7 gene, its RNA or protein products, or anothermolecular entity whose activity or expression impinges upon the activityor expression of SMAD7 or its products either exclusively or with a highdegree of specificity. In the case of antibody inhibitors of SMAD7,specificity can be engineered into the antibody via inclusion of proteinsequences known to bind SMAD7 protein epitopes with a high degree ofspecificity. In the case of small molecule inhibitors of SMAD7, chemicalgroups can be included in the formulation of the small molecule thatallow binding to specific features of SMAD7 protein. Antisenseoligonucleotides can be designed such that the targeting portion of theincorporated nucleotide sequence of each antisense oligonucleotide iscompletely or almost completely complementary to the SMAD7 mRNAsequence. Incorporation of such complementary or nearly complementarynucleotide sequences allows one to engineer antisense oligonucleotideswith a high degree of specificity for a given target. Specificity can beassessed via measurement of parameters such as dissociation constant, orother criteria such as changes in protein or RNA expression levels orother assays that measure SMAD7 activity or expression.

Specific SMAD7 inhibitors can include, for example, small bindingmolecules, e.g., natural and synthetic compounds, antibodies, aptamers,intramers, RNAi (double stranded RNA, siRNA) and anti-SMAD7 antisensemolecules for treating colorectal cancer and/or inhibiting colorectalcancer cell growth. SMAD7 inhibitors may also comprise truncated and/ormutated SMAD7 molecules which interfere with SMAD7 activity, bindingpartners, or substrates and which, thereby, inhibit SMAD7 function.

“Effective amount,” as used herein, refers to the amount of an agentthat is sufficient to at least partially treat a condition whenadministered to a patient. The therapeutically effective amount willvary depending on the condition, the route of administration of thecomponent, and the age, weight, etc. of the patient being treated.Accordingly, an effective amount of a specific inhibitor of SMAD7 is theamount of inhibitor necessary to treat colorectal cancer in a patientsuch that administration of the agent prevents the colorectal cancerfrom occurring in a subject, prevents colorectal cancer progression(e.g., prevents onset of events such as tumorigenesis, tumor growth, ormetastasis), or relieves or completely ameliorates all associatedsymptoms of the colorectal cancer, i.e. causes regression of thedisease.

The present invention also provides a method for treating colorectalcancer via administration of a pharmaceutical composition comprising anantisense oligonucleotide against SMAD7. In another aspect, theinvention provides a pharmaceutical composition for use in treatingcolorectal cancer. The pharmaceutical composition may be comprised of aninhibitor of SMAD7, such as an antisense oligonucleotide that targetsSMAD7, and a pharmaceutically acceptable carrier. As used herein theterm “pharmaceutical composition” means, for example, a mixturecontaining a specified amount of a therapeutic compound, e.g. atherapeutically effective amount, of a therapeutic compound in apharmaceutically acceptable carrier to be administered to a mammal,e.g., a human, in order to treat colorectal cancer. In some embodiments,contemplated herein are pharmaceutical compositions comprising acontemplated antisense oligonucleotide against SMAD7 and apharmaceutically acceptable carrier. In another aspect, the inventiondiscloses use of an antisense oligonucleotide against SMAD7 in themanufacture of a medicament for treating colorectal cancer.“Medicament,” as used herein, has essentially the same meaning as theterm “pharmaceutical composition.”

As used herein, “pharmaceutically acceptable carrier” means buffers,carriers, and excipients suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio. The carrier(s) should be “acceptable” inthe sense of being compatible with the other ingredients of theformulations and not deleterious to the recipient. Pharmaceuticallyacceptable carriers include buffers, solvents, dispersion media,coatings, isotonic and absorption delaying agents, and the like, thatare compatible with pharmaceutical administration. The use of such mediaand agents for pharmaceutically active substances is known in the art.In one embodiment the pharmaceutical composition is administered orallyand includes an enteric coating suitable for regulating the site ofabsorption of the encapsulated substances within the digestive system orgut. For example, an enteric coating can include anethylacrylate-methacrylic acid copolymer.

In one embodiment, a contemplated antisense oligonucleotide againstSMAD7 and any pharmaceutical composition thereof may be administered byone or several routes, including orally, topically, parenterally, e.g.,subcutaneous injection, by inhalation spray, or rectally. The termparenteral as used herein includes subcutaneous injections,intrapancreatic administration, intravenous, intramuscular,intraperitoneal, intrasternal injection or infusion techniques. Forexample, the antisense oligonucleotide against SMAD7 may be administeredsubcutaneously to a subject. In another example, the antisenseoligonucleotide against SMAD7 may be administered orally to a subject.In another example, the antisense oligonucleotide against SMAD7 may beadministered directly to a colorectal tumor or colorectal cancer cellsvia parenteral administration.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A shows SMAD7 immunostaining in non-tumoral (NT) and tumoral (T)areas of a patient with sporadic colorectal cancer; FIG. 1B is a Westernblot showing SMAD7 and β-actin levels in NT and T tissue from twopatients with sporadic colorectal cancer; FIG. 1C is a Western blotshowing SMAD7 and β-actin levels in IECs and DLD-1 and HCT-116 cells;and FIG. 1D is a Western blot showing expression levels of SMAD7 andβ-actin in four colorectal cancer cell lines (HCT-116, HCT-115, HT-29,and DLD-1) and one hepatocellular carcinoma cell line (HepG2).

FIG. 2A is a series of dot-plots depicting the total number of cells andindicating percentages of propidium iodide (PI)- and fluorescent(FITC)-positive HCT-116 cells transfected with either unlabeled SMAD7sense oligonucleotide or with increasing doses of the FITC-conjugatedSMAD7 antisense oligonucleotide GED-0301(GED-0301 FITC-conjugated); FIG.2B is a Western blot showing SMAD7 and β-actin levels in HCT-116 cellsfollowing transfection with either SMAD7 sense or GED-0301oligonucleotides; FIG. 2C depicts a graph showing the percent ofproliferating HCT-116 or DLD-1 cells following no treatment (Untr) ortransfection with SMAD7 sense or GED-0301 oligonucleotides (left) andhistograms showing the percent of proliferating HCT-116 cells followingtransfection with SMAD7 sense (top) or GED-0301 (bottom)oligonucleotides; and FIG. 2D is a graph showing the percent of HCT-116cells in different cell cycle phases following no treatment (Untr) ortransfection with SMAD7 sense or GED-0301 oligonucleotides.

FIG. 3 is a Western blot showing expression levels in HCT-116 cells ofCDK2 phosphorylated at Threoninine-14 or Tyrosine-15 (p-CDK2(Thr-14/Tyr-15)), total CDK2 (CDK2), and β-actin following nostimulation (Uns) or transfection with SMAD7 sense or GED-0301 (SMAD7AS) oligonucleotides.

FIG. 4A shows graphs depicting the percent of HCT-116 cell death at 24(top) or 48 hours (bottom) following no treatment (Untr) or transfectionwith either SMAD7 sense or GED-0301 oligonucleotides and dot-plots(bottom) illustrating PI and Annexin V (AV) staining followingtransfection of SMAD7 sense or GED-0301 oligonucleotides; FIG. 4B showsa series of dot-plots quantifying active caspase-3 in HCT-116 cells atdifferent time points following no treatment (Untr) or transfection ofSMAD7 sense or GED-0301 oligonucleotides; FIG. 4C is a graph showing thepercent of cells exposed toN-(2-Quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)methyl ketone(Q-VD-OPH) with active caspase-3 after no treatment or transfection withSMAD7 sense or GED-0301 oligonucleotides; FIG. 4D is a graph showing thepercent of cell death in cells exposed to Q-VD-OPH and then exposed tono treatment or transfection with SMAD7 sense or GED-0301oligonucleotides; and FIG. 4E is a graph showing the percent ofproliferating cells exposed to Q-VD-OPH followed by either no treatmentor transfection with SMAD7 sense or GED-0301 oligonucleotides.

FIG. 5A shows SMAD7 immunostaining in non-tumoral or tumoral tissue froma mouse following azoxymethane and dextran sulfate sodium (AOM+DSS)treatment, and FIG. 5B is a graph showing relative SMAD7 mRNA expressionin non-tumoral and tumoral tissue from mice following AOM+DSS treatment.

FIG. 6 shows hematoxylin and eosin (H&E) staining and Proliferating CellNuclear Antigen (PCNA) immunostaining of colorectal cancer tissueexplants transfected with either SMAD7 sense or GED-0301oligonucleotides.

FIG. 7A shows distribution of FITC signal in HCT-116-derived xenograftsexposed to a single injection of either PBS (CTR) or FITC-conjugatedGED-0301 after injection into HCT-116-colonized Rag1^(−/−) mice; FIG. 7Bis a Western blot showing SMAD7 and β-actin expression in proteinextracts of HCT-116-derived xenografts exposed to either SMAD sense orGED-0301 oligonucleotides; FIG. 7C is a graph quantifying tumor volumeof xenografts derived from mice treated with either SMAD7 sense orGED-0301 oligonucleotides (left) and a representative photograph ofxenografts from the same experiment; and FIG. 7D shows PCNAimmunostaining of xenografts treated with either SMAD7 sense or GED-0301oligonucleotides.

DETAILED DESCRIPTION Colorectal Cancer

The present invention provides methods for treatment of colorectalcancer. “Colorectal cancer,” as used herein, refers to a diseasecharacterized by unchecked proliferation of cells of the largeintestine, including cells of the colon or rectum. Colorectal cancertypically originates in epithelial cells of the large intestine withintestinal crypt stem cells being a likely cell of origin. Geneticmutations that result in carcinogenesis include mutations in members ofthe Wnt signaling pathway such as β-catenin, APC, AXIN1, AXIN2, TCF7L2,and NKD1, members of the TGF-β cell signaling pathway such as TGF-β1 andSMAD family members, proteins that regulate the balance between cellproliferation and cell death such as TP53, and other proteins such asDCC. Proliferation of the epithelial cells that carry the causativemutation or mutations can result in invasive growth into the musclelayers and through the bowel wall. Symptoms of colorectal cancertypically include rectal bleeding, anemia, constipation, blood in thestool, weight loss, fever, loss of appetite, and nausea or vomiting. Thevast majority of colorectal cancer tumors can be classified asadenocarcinomas while lymphomas and squamous cell carcinomas areobserved in a smaller subset of cases. Accordingly, the term “colorectaltumor,” as used herein, refers to any abnormal malignant growth oftissue associated with cells originating in the large intestine orcolorectal cancer pathology.

The term “colorectal cancer cells,” as used herein, refers to any cellof origin giving rise to colorectal cancer or a colorectal tumor, a cellassociated with the tumorigenesis, growth, evolution, maintenance, orsupport of a colorectal tumor, or any other cell associated with thepathological manifestation of colorectal cancer. “Growth of colorectalcancer cells,” as used herein, refers to the unchecked or abnormalproliferation of cells associated with a colorectal cancer cell oforigin, a colorectal tumor cell, or any cell associated with themanifestation of colorectal cancer pathology. Growth of colorectalcancer cells may be the result of abnormal cell cycle activity, failureto induce cell cycle checkpoints, failure to induce apoptosis, or lossof other tumor suppressor activities.

Treatment and Evaluation

The terms “treat”, “treatment”, “treating” and the like are used hereinto generally mean obtaining a desired pharmacological and/orphysiological effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of partially or completely curing a diseaseand/or adverse effect attributed to the disease. The term “treatment” asused herein covers any treatment of a disease in a mammal, particularlya human, and includes: (a) preventing the disease from occurring in asubject which may be predisposed to the disease but has not yet beendiagnosed as having it; (b) inhibiting the disease, i.e. arresting itsdevelopment; or (c) relieving the disease, i.e. causing regression ofthe disease.

Efficacy of treatment may be evaluated by means of evaluation of grosssymptoms associated with colorectal cancer, analysis of tissuehistology, biochemical assay, imaging methods such as, for example,magnetic resonance imaging, or other known methods. For instance,efficacy of treatment may be evaluated by analyzing anemic state, rectalbleeding, tumor size, or other aspects of gross pathology associatedwith colorectal cancer following administration of a SMAD7 inhibitor toa colorectal cancer patient. Efficacy of treatment may also be evaluatedat the tissue or cellular level, for example, by means of obtaining atissue or tumor biopsy and evaluating gross tissue or cell morphology orstaining properties. Biochemical assays that examine protein or RNAexpression may also be used to evaluate efficacy of treatment. Forinstance, one may evaluate PCNA, p-CDK2 (Thr-14/Tyr-15), or levels ofanother protein indicative of cell proliferation or cell death activityin dissociated cells or non-dissociated tissue via immunocytochemical,immunohistochemical, or Western blotting methods. One may also evaluatethe presence or level of expression of useful biomarkers found in plasmaor tumoral or non-tumoral tissue to evaluate cancer progression andefficacy of treatment.

In evaluating efficacy of treatment, suitable controls may be chosen toensure a valid assessment. For instance, one can compare symptomsevaluated in a patient with colorectal cancer following administrationof an inhibitor of SMAD7 to those symptoms in the same patient prior totreatment or in another patient not diagnosed with colorectal cancer.Alternatively, one may compare the results of biochemical orhistological analysis of tumoral tissue following administration of aSMAD7 inhibitor with those of non-tumoral tissue from the same patientor from an individual not diagnosed with colorectal cancer or from thesame patient prior to administration of the SMAD7 inhibitor.

Validation of SMAD7 inhibition may be determined by direct or indirectassessment of SMAD7 expression levels or activity. For instance,biochemical assays that measure SMAD7 protein or RNA expression may beused to evaluate overall SMAD7 inhibition. For instance, one may measureSMAD7 protein levels in tumor tissue by Western blot to evaluate overallSMAD7 levels. One may also measure SMAD7 mRNA levels by means ofNorthern blot or quantitative polymerase chain reaction to determineoverall SMAD7 inhibition. One may also evaluate SMAD7 protein levels orlevels of another protein indicative of SMAD7 activity in dissociatedcells or non-dissociated tissue via immunocytochemical orimmunohistochemical methods. SMAD7 inhibition may also be evaluatedindirectly by measuring parameters such as cell cycle phasedistribution, staining with markers of cell death such as Annexin V orCaspase III, or measuring alteration in other parameters correlated withchanges in SMAD7 activity. For instance, one may measure levels ofactive caspase-3 in cells of a tumor treated with a SMAD7 inhibitor asan indication of SMAD7 activity in said cells. One may also evaluate thepresence or level of expression of useful biomarkers found in plasma ortumoral or non-tumoral tissue to evaluate efficacy of SMAD7 inhibition.

In evaluating efficacy of SMAD7 knockdown, suitable controls may bechosen to ensure a valid assessment. For instance, one may compare theresults of biochemical or histological analysis of tumoral tissuefollowing administration of a SMAD7 inhibitor with those of non-tumoraltissue from the same patient or from an individual not diagnosed withcolorectal cancer or from the same patient prior to administration ofthe SMAD7 inhibitor.

A “patient,” as described herein, refers to any animal at risk for orsuffering from colorectal cancer, including, but not limited to,mammals, primates, and humans. For example, a patient may be anindividual diagnosed with a high risk of colorectal cancer developmentor someone who has been diagnosed with colorectal cancer. In certainembodiments, the patient may be a non-human mammal such as, for example,a cat, a dog, or a horse.

Inhibitors of SMAD7

In certain embodiments, an anti-SMAD7 antisense oligonucleotide maytarget site 403, 233, 294, 295, 296, 298, 299, and/or 533 (i.e.,nucleotides 403, 233, 294, 295, 296, 298, 299, and 533, respectively) ofthe human SMAD7 mRNA. In an exemplary embodiment, the anti-SMAD7antisense oligonucleotide targets nucleic acids 403-423 of human SMAD7mRNA. Exemplary SMAD7 inhibitors include those disclosed in PCTPublication No. WO2010/054826, which is hereby incorporated by referencein its entirety.

In certain embodiments, an antisense oligonucleotide may be derived fromthe following anti-SMAD7 antisense oligonucleotide5′-GTCGCCCCTTCTCCCCGCAGC-3′ (SEQ ID NO: 3).

It is contemplated herein that an antisense oligonucleotide targetingSMAD7 may comprise a mixed-backbone wherein the cytosine residues in aCpG pair are replaced by 5′-methylcytosine (abbreviated as Me-dC).Methylphosphonate linkages may also be placed at the 5′ and/or 3′ endsof an antisense oligonucleotide (abbreviated as MeP). The phosphatebackbone of a contemplated anti-SMAD7 antisense oligonucleotide mayoptionally include 1, 2, 3, 4 or more phosphorothioate bonds (e.g.,phosphorothioate bonds would replace phosphodiester bonds). In anembodiment, all phosphate bonds may be phosphorothioate bonds.

Exemplary antisense oligonucleotide therapies that target SMAD7 include,but are not limited to:

5′-GTXYCCCCTTCTCCCXYCAG-3′ (SEQ ID NO: 4), wherein X is a nucleotidecomprising a nitrogenous base selected from the group consisting ofcytosine and 5-methylcytosine or a 2′-O-methylcytosine nucleoside, andwherein Y is a nucleotide comprising a nitrogenous base selected fromthe group consisting of guanine and 5-methylguanine or a2′-O-methylguanine nucleoside, provided that at least one of thenucleotides X or Y comprises a methylated nitrogenous base;

5′-GTXGCCCCTTCTCCCXGCAG-3′ (SEQ ID NO: 5), wherein X is 5-methyl2′-deoxycytidine 5′-monophosphate;

5′-GTXGCCCCTTCTCCCXGCAGC-3′ (SEQ ID NO: 6), wherein X is 5-methyl2′-deoxycytidine 5′-monophosphate;

5′-ZTXGCCCCTTCTCCCXGCAZ-3′ (SEQ ID NO: 7), wherein X is 5-methyl2′-deoxycytidine 5′-monophosphate and Z is 2′-deoxyguanosinemethylphosphonate;

5′-ZTXGCCCCTTCTCCCXGCAZC-3′ (SEQ ID NO: 8), wherein X is 5-methyl2′-deoxycytidine 5′-monophosphate and Z is 2′-deoxyguanosinemethylphosphonate.

In a particular embodiment, contemplated SMAD7 antisense may be asequence comprising one of:

5′-GTXGCCCCTTCTCCCXGCAG-3′ (SEQ ID NO: 9), wherein X is 5-methyl2′-deoxycytidine 5′-monophosphorothioate;

5′-GTXGCCCCTTCTCCCXGCAGC-3′ (SEQ ID NO: 10), wherein X is 5-methyl2′-deoxycytidine 5′-monophosphorothioate;

5′-ZTXGCCCCTTCTCCCXGCAZ-3′ (SEQ ID NO: 11), wherein X is 5-methyl2′-deoxycytidine 5′-monophosphate and Z is 2′-deoxyguanosinemethylthiophosphonate;

5′-ZTXGCCCCTTCTCCCXGCAZC-3′ (SEQ ID NO: 12), wherein X is 5-methyl2′-deoxycytidine 5′-monophosphate and Z is 2′-deoxyguanosinemethylthiophosphonate.

For example, SEQ ID NOs. 9-12 include 1, 2, 3, 4 or morephosphorothioate bonds. In an embodiment, all O,O phosphonate bonds ofSEQ ID NOs. 9-12 are phosphorothioate bonds.

Pharmaceutical Compositions and Routes of Administration

Pharmaceutical compositions containing an antisense oligonucleotideagainst SMAD7, such as those disclosed herein, can be presented in adosage unit form and can be prepared by any suitable method. Apharmaceutical composition should be formulated to be compatible withits intended route of administration. Useful formulations can beprepared by methods well known in the pharmaceutical art. For example,see Remington's Pharmaceutical Sciences, 18th ed. (Mack PublishingCompany, 1990).

Pharmaceutical formulations preferably are sterile. Sterilization can beaccomplished, for example, by filtration through sterile filtrationmembranes. Where the composition is lyophilized, filter sterilizationcan be conducted prior to or following lyophilization andreconstitution.

Parenteral Administration

The pharmaceutical compositions of the invention can be formulated forparenteral administration, e.g., formulated for injection via theintravenous, intramuscular, subcutaneous, intralesional, orintraperitoneal routes. The preparation of an aqueous composition, suchas an aqueous pharmaceutical composition containing a SMAD7 inhibitor,will be known to those of skill in the art in light of the presentdisclosure. Typically, such compositions can be prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forusing to prepare solutions or suspensions upon the addition of a liquidprior to injection can also be prepared; and the preparations can alsobe emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Solutions of active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. In addition, sterile, fixed oils may be employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid can be used in the preparation ofinjectables. The sterile injectable preparation may also be a sterileinjectable solution, suspension, or emulsion in a nontoxic parenterallyacceptable diluent or solvent, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. In one embodiment, the SMAD7 inhibitor may besuspended in a carrier fluid comprising 1% (w/v) sodiumcarboxymethylcellulose and 0.1% (v/v) TWEEN™ 80. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents.Generally, dispersions are prepared by incorporating the varioussterilized active ingredients into a sterile vehicle which contains thebasic dispersion medium and the required other ingredients from thoseenumerated above. Sterile injectable solutions of the invention may beprepared by incorporating a SMAD7 inhibitor in the required amount ofthe appropriate solvent with various of the other ingredients enumeratedabove, as required, followed by filtered sterilization. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The injectable formulations can be sterilized, forexample, by filtration through a bacteria-retaining filter.

The preparation of more, or highly, concentrated solutions forintramuscular injection is also contemplated. In this regard, the use ofDMSO as solvent is preferred as this will result in extremely rapidpenetration, delivering high concentrations of the SMAD7 inhibitor to asmall area.

Suitable preservatives for use in such a solution include benzalkoniumchloride, benzethonium chloride, chlorobutanol, thimerosal and the like.Suitable buffers include boric acid, sodium and potassium bicarbonate,sodium and potassium borates, sodium and potassium 10 carbonate, sodiumacetate, sodium biphosphate and the like, in amounts sufficient tomaintain the pH at between about pH 6 and pH 8, and preferably, betweenabout pH 7 and pH 7.5. Suitable tonicity agents are dextran 40, dextran70, dextrose, glycerin, potassium chloride, propylene glycol, sodiumchloride, and the like, such that the sodium chloride equivalent of theophthalmic solution is in the range 0.9 plus or minus 0.2%. Suitableantioxidants and stabilizers include sodium bisulfite, sodiummetabisulfite, sodium thiosulfite, thiourea and the like. Suitablewetting and clarifying agents include polysorbate 80, polysorbate 20,poloxamer 282 and tyloxapol. Suitable viscosity-increasing agentsinclude dextran 40, dextran 70, gelatin, glycerin,hydroxyethylcellulose, hydroxymethylpropylcellulose, lanolin,methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol,polyvinylpyrrolidone, carboxymethylcellulose and the like.

In an exemplary embodiment, a pharmaceutical composition forsubcutaneous administration of an antisense oligonucleotide againstSMAD7 comprises an antisense oligonucleotide such as that represented bySEQ ID NO: 6, or a pharmaceutically acceptable salt thereof (such as asodium salt), and a pharmaceutically acceptable carrier.

Oral Administration

In some embodiments, contemplated herein are compositions suitable fororal delivery of an antisense oligonucleotide, e.g., tablets, thatinclude an enteric coating, e.g., a gastro-resistant coating, such thatthe compositions may deliver the antisense compound to, e.g., the colonof a patient. For example, such administration may result in a topicaleffect, substantially topically applying the antisense compound directlyto an affected portion of the colon of a patient. Such administration,may, in some embodiments, substantially avoid unwanted systemicabsorption of the antisense compound.

For example, a tablet for oral administration is provided that comprisesgranules (e.g., is at least partially formed from granules) that includea disclosed antisense compound, e.g., GED-0301, and pharmaceuticallyacceptable excipients. Such a tablet may be coated with an entericcoating. Contemplated tablets may include pharmaceutically acceptableexcipients such as fillers, binders, disintegrants, and/or lubricants,as well as coloring agents, release agents, coating agents, sweetening,flavoring such as wintergreen, orange, xylitol, sorbitol, fructose, andmaltodextrin, and perfuming agents, preservatives and/or antioxidants.

In some embodiments, contemplated pharmaceutical formulations include anintra-granular phase that includes a contemplated antisense compound,e.g. GED-0301, or a pharmaceutically acceptable salt, e.g., GED-0301 anda pharmaceutically acceptable filler. For example, GED-0301 and a fillermay be blended together, optionally, with other excipients, and formedinto granules. In some embodiments, the intragranular phase may beformed using wet granulation, e.g. a liquid (e.g., water) is added tothe blended antisense compound and filler, and then combination isdried, milled and/or sieved to produce granules. One of skill in the artwould understand that other processes may be used to achieve anintragranular phase.

In some embodiments, contemplated formulations include an extra-granularphase, which may include one or more pharmaceutically acceptableexcipients, and which may be blended with the intragranular phase toform a disclosed formulation.

A disclosed formulation may include an intragranular phase that includesa filler. Exemplary fillers include, but are not limited to, cellulose,gelatin, calcium phosphate, lactose, sucrose, glucose, mannitol,sorbitol, microcrystalline cellulose, pectin, polyacrylates, dextrose,cellulose acetate, hydroxypropylmethyl cellulose, partiallypregelatinized starch, calcium carbonate, and others includingcombinations thereof.

In some embodiments, a disclosed formulation may include a intragranularphase and/or a extragranular phase that includes a binder, which maygenerally function to hold the ingredients of the pharmaceuticalformulation together. Exemplary binders of the invention may include,but are not limited to, the following: starches, sugars, cellulose ormodified cellulose such as hydroxypropyl cellulose, lactose,pregelatinized maize starch, polyvinyl pyrrolidone, hydroxypropylcellulose, hydroxypropylmethyl cellulose, low substituted hydroxypropylcellulose, sodium carboxymethyl cellulose, methyl cellulose, ethylcellulose, sugar alcohols and others including combinations thereof.

Contemplated formulations, e.g., that include an intragranular phaseand/or an extragranular phase, may include a disintegrant such as butare not limited to, starch, cellulose, crosslinked polyvinylpyrrolidone, sodium starch glycolate, sodium carboxymethyl cellulose,alginates, corn starch, crosmellose sodium, crosslinked carboxymethylcellulose, low substituted hydroxypropyl cellulose, acacia, and othersincluding combinations thereof. For example, an intragranular phaseand/or an extragranular phase may include a disintegrant.

In some embodiments, a contemplated formulation includes anintra-granular phase comprising a disclosed antisense compound andexcipients chosen from: mannitol, microcrystalline cellulose,hydroxypropylmethyl cellulose, and sodium starch glycolate orcombinations thereof, and an extra-granular phase comprising one or moreof: microcrystalline cellulose, sodium starch glycolate, and magnesiumstearate or mixtures thereof.

In some embodiments, a contemplated formulation may include a lubricant,e.g. an extra-granular phase may contain a lubricant. Lubricants includebut are not limited to talc, silica, fats, stearin, magnesium stearate,calcium phosphate, silicone dioxide, calcium silicate, calciumphosphate, colloidal silicon dioxide, metallic stearates, hydrogenatedvegetable oil, corn starch, sodium benzoate, polyethylene glycols,sodium acetate, calcium stearate, sodium lauryl sulfate, sodiumchloride, magnesium lauryl sulfate, talc, and stearic acid.

In some embodiments, the pharmaceutical formulation comprises an entericcoating. Generally, enteric coatings create a barrier for the oralmedication that controls the location at which the drug is absorbedalong the digestive track. Enteric coatings may include a polymer thatdisintegrates a different rates according to pH. Enteric coatings mayinclude for example, cellulose acetate phthalate, methylacrylate-methacrylic acid copolymers, cellulose acetate succinate,hydroxylpropylmethyl cellulose phthalate, methylmethacrylate-methacrylic acid copolymers, ethylacrylate-methacrylic acidcopolymers, methacrylic acid copolymer type C, polyvinylacetate-phthalate, and cellulose acetate phthalate.

Exemplary enteric coatings include Opadry® AMB, Acryl-EZE®, Eudragit®grades. In some embodiments, an enteric coating may comprise about 5% toabout 10%, about 5% to about 20%, 8 to about 15%, about 8% to about 18%,about 10% to about 12%, or about 12 to about 16%, of a contemplatedtablet by weight. For example, enteric coatings may include anethylacrylate-methacrylic acid copolymer.

For example, a tablet is provided that comprises or consists essentiallyof about 0.5% to about 70%, e.g. about 0.5% to about 10%, or about 1% toabout 20%, by weight of an antisense oligonucleotide or apharmaceutically acceptable salt thereof (e.g. GED-0301). Such a tabletmay include for example, about 0.5% to about 60% by weight of mannitol,e.g. about 30% to about 50% by weight mannitol, e.g. about 40% by weightmannitol; and/or about 20% to about 40% by weight of microcrystallinecellulose, or about 10% to about 30% by weight of microcrystallinecellulose. For example, a disclosed tablet may comprise an intragranularphase that includes about 30% to about 60%, e.g. about 45% to about 65%by weight, or alternatively, about 5 to about 10% by weight GED-0301,about 30% to about 50%, or alternatively, about 5% to about 15% byweight mannitol, about 5% to about 15% microcrystalline cellulose, about0% to about 4%, or about 1% to about 7% hydroxypropylmethylcellulose,and about 0% to about 4%, e.g. about 2% to about 4% sodium starchglycolate by weight.

In another embodiment, a pharmaceutical tablet formulation for oraladministration of an antisense oligonucleotide against SMAD7 comprisesan intra-granular phase, wherein the intra-granular phase includes anantisense oligonucleotide such as GED-0301, or a pharmaceuticallyacceptable salt thereof (such as a sodium salt), and a pharmaceuticallyacceptable filler, and which may also include an extra-granular phase,that may include a pharmaceutically acceptable excipient such as adisintegrant. The extra-granular phase may include components chosenfrom microcrystalline cellulose, magnesium stearate, and mixturesthereof. The pharmaceutical composition may also include an entericcoating of about 12% to 16% by weight of the tablet. For example, apharmaceutically acceptable tablet for oral use may comprise about 0.5%to 10% by weight of an antisense oligonucleotide, e.g., GED-0301, or apharmaceutically acceptable salt thereof, about 30% to 50% by weightmannitol, about 10% to 30% by weight microcrystalline cellulose, and anenteric coating comprising an ethylacrylate-methacrylic acid copolymer.

In another example, a pharmaceutically acceptable tablet for oral usemay comprise an intra-granular phase, comprising about 5 to about 10% byweight of an antisense oligonucleotide, e.g., GED-0301, or apharmaceutically acceptable salt thereof, about 40% by weight mannitol,about 8% by weight microcrystalline cellulose, about 5% by weighthydropropylmethyl cellulose, and about 2% by weight sodium starchglycolate; an extra-granular phase comprising about 17% by weightmicrocrystalline cellulose, about 2% by weight sodium starch glycolate,about 0.4% by weight magnesium stearate; and an enteric coating over thetablet comprising an ethylacrylate-methacrylic acid copolymer.

In some embodiments the pharmaceutical composition may contain anenteric coating comprising about 13% or about 15%, 16%, 17% or 18% byweight, e.g., AcyrlEZE® (see, e.g., PCT Publication No. WO2010/054826,which is hereby incorporated by reference in its entirety).

The rate at which point the coating dissolves and the active ingredientis released is its dissolution rate. In an embodiment, a contemplatedtablet may have a dissolution profile, e.g. when tested in a USP/EP Type2 apparatus (paddle) at 100 rpm and 37° C. in a phosphate buffer with apH of 7.2, of about 50% to about 100% of the oligonucleotide releasingafter about 120 minutes to about 240 minutes, for example after 180minutes. In another embodiment, a contemplated tablet may have adissolution profile, e.g. when tested in a USP/EP Type 2 apparatus(paddle) at 100 rpm and 37° C. in diluted HCl with a pH of 1.0, wheresubstantially none of the oligonucleotide is released after 120 minutes.A contemplated tablet, in another embodiment, may have a dissolutionprofile, e.g. when tested in USP/EP Type 2 apparatus (paddle) at 100 rpmand 37° C. in a phosphate buffer with a pH of 6.6, of about 10% to about30%, or not more than about 50%, of the oligonucleotide releasing after30 minutes.

Disclosed formulations, e.g. tablets, in some embodiments, when orallyadministered to the patient may result in minimal plasma concentrationof the oligonucleotide in the patient. In another embodiment, disclosedformulations, when orally administered to a patient, topically deliverto the colon or rectum of a patient, e.g. to an affected or diseasedsite of a patient.

In some embodiments, methods provided herein may further includeadministering at least one other agent that is directed to treatment ofdiseases and disorders disclosed herein. In one embodiment, contemplatedother agents may be co-administered (e.g., sequentially orsimultaneously).

Agents contemplated include immunosuppressive agents includingglucocorticoids, cytostatics, antibodies, agents acting onimmunophilins, interferons, opioids, TNF binding proteins,mycophenolate, and small biological agents. For example, contemplatedimmunosuppressive agents include, but are not limited to: tacrolimus,cyclosporine, pimecrolimus, sirolimus, everolimus, mycophenolic acid,fingolimod, dexamethasone, fludarabine, cyclophosphamide, methotrexate,azathioprine, leflunomide, teriflunomide, anakinra, anti-thymocyteglobulin, anti-lymphocyte globulin, muromonab-CD3, afutuzumab,rituximab, teplizumab, efalizumab, daclizumab, basiliximab, adalimumab,infliximab, and etanercept.

Dosage and Frequency of Administration

Exemplary formulations include dosage forms that include or consistessentially of about 35 mg to about 500 mg of an antisenseoligonucleotide against SMAD7. For example, formulations that includeabout 35 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg,120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg,or 250 mg of an antisense oligonucleotide against SMAD7 are contemplatedherein. In one embodiment, a formulation may include about 40 mg, 80 mg,or 160 mg of an antisense oligonucleotide against SMAD7. In someembodiments, a formulation may include at least 100 μg of an antisenseoligonucleotide against SMAD7. For example, formulations may includeabout 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 15 mg,20 mg, or 25 mg of an antisense oligonucleotide against SMAD7. Theamount administered will depend on variables such as the type and extentof disease or indication to be treated, the overall health and size ofthe patient, the in vivo potency of the antisense oligonucleotide, thepharmaceutical formulation, and the route of administration. The initialdosage can be increased beyond the upper level in order to rapidlyachieve the desired blood-level or tissue level. Alternatively, theinitial dosage can be smaller than the optimum, and the dosage may beprogressively increased during the course of treatment. Human dosage canbe optimized, e.g., in a conventional Phase I dose escalation studydesigned to run from 40 mg to 160 mg. Dosing frequency can vary,depending on factors such as route of administration, dosage amount andthe disease being treated. Exemplary dosing frequencies are once perday, once per week and once every two weeks. In some embodiments, dosingis once per day for 7 days.

EXAMPLES

The invention is further illustrated by the following examples. Theexamples are provided for illustrative purposes only, and are not to beconstrued as limiting the scope or content of the invention in any way.

Example 1: SMAD7 Protein Expression Levels in Colorectal Cancer Cells

SMAD7 protein expression levels were evaluated in paired colorectalcancer tumoral and non-tumoral colonic mucosal specimens taken from 6patients undergoing colonic resection for colorectal cancer. SMAD7immunostaining of tumoral (T) and non-tumoral (NT) tissue revealed amarked accumulation of SMAD7 protein in tumoral as compared to thenon-tumoral tissue (FIG. 1A, representative of three separateexperiments in which sections of six patients with colorectal cancerwere analyzed). No staining was observed when colorectal cancer sectionswere incubated with isotype control IgG (not shown). Additionally, SMAD7protein levels in tumoral (T) and non-tumoral (NT) tissue were evaluatedby Western blotting in two colorectal cancer patients. Western blottingof cell extract demonstrated observably higher levels of SMAD7 intumoral tissue compared to non-tumoral tissue (FIG. 1B). β-actin wasused as a loading control.

SMAD7 protein expression was also investigated in colorectal cancer celllines. Total protein extracts were collected from cells of thecolorectal cancer cell lines DLD-1 and HCT-116 as well as from normalcolonic epithelial cells (IECs) and evaluated for SMAD7 expression byWestern blotting (FIG. 1C). IECs were isolated from the macroscopicallyand microscopically unaffected mucosa of patients undergoing colectomyfor sporadic colorectal cancer. Western blotting revealed markedlyhigher expression of SMAD7 protein in DLD-1 and HCT-116 cells comparedto IECs. Additionally, Western blot detection of SMAD7 protein inextracts collected from a series of colorectal cancer cell lines,including HCT-116, HT-115, HT-29, and DLD-1, revealed high levels ofSMAD7 expression (FIG. 1D). In the same experiment, SMAD7 expression wasevaluated in HepG2 cells, a hepatocellular carcinoma cell line known toexpress high levels of SMAD7 protein. β-actin was used as a loadingcontrol in both sets of experiments.

Example 2: Knockdown of Colorectal Cancer Cell SMAD7 Protein Levels bySMAD7 Antisense Oligonucleotide

The transfection of cancer cells with the SMAD7 antisenseoligonucleotide GED-0301 (SEQ ID NO: 6) was evaluated in cells of theHCT-116 cell line. HCT-116 cells were transfected with either unlabeledsense (SMAD7 sense) oligonucleotide or with increasing doses (0.5 μg/ml,1 μg/ml, or 2 μg/ml) of FITC-conjugated GED-0301 for six hours. FIG. 2Ashows representative dot-plots quantifying the percentages ofPI-positive and FITC-positive transfected HCT-116 cells. Hightransfection efficiency was achieved with very low levels of cell deathas evaluated by PI and FITC staining. One of two representativeexperiments in which similar results were obtained is shown in FIG. 2A.

Knockdown of SMAD7 protein levels following transfection of varyingamounts of GED-0301 was also evaluated in HCT-116 cells. HCT-116 cellswere transfected either with SMAD7 sense oligonucleotide (at 2 μg/ml) orGED-0301 (at 0.5 μg/ml, 1 μg/ml, or 2 μg/ml) for twelve hours. Cellswere subsequently washed with phosphate buffered saline (PBS), culturedwith fresh medium for six hours, washed again with PBS, and cultured foran additional 24 hours. SMAD7 and β-actin levels were then analyzed byWestern blotting. FIG. 2B shows one of three representative experiments,demonstrating observable knockdown of SMAD7 protein levels in cellstransfected with increasing amounts of GED-0301 as compared to cellstransfected with SMAD7 sense oligonucleotide. These results demonstratedthat a SMAD7 antisense oligonucleotide could be transfected intocolorectal cancer cells and could induce robust knockdown of SMAD7protein levels.

Example 3: SMAD7 Antisense Oligonucleotide Administration Affects CellCycle Dynamics in Colorectal Cancer Cells

Cell proliferation was assessed in HCT-116 and DLD-1 cells followingadministration of SMAD7 sense and GED-0301 oligonucleotides. HCT-116 andDLD-1 cells were either not transfected (Untr) or transfected with SMAD7sense or GED-0301 oligonucleotides at 1 μg/ml. Twelve hourspost-transfection, cells were washed with PBS, cultured for six hoursmore, re-washed with PBS, and labeled with carboxyfluorescein diacetatesuccinimidyl ester (CFSE) for 30 minutes. Labeled cells were then washedwith PBS and re-cultured in fresh medium for an additional 24 hours. Thepercentage of proliferating cells was evaluated by flow-cytometry. Asignificant decrease in cell proliferation was observed in both DLD-1(white bars) and HCT-116 (black bars) cells transfected with GED-0301 ascompared to cells transfected with SMAD7 sense oligonucleotide (FIG. 2C;HCT-116: SMAD7 sense-transfected cells vs GED-0301-transfected cells,*P<0.001; DLD-1: SMAD7 sense-transfected cells vs GED-0301-transfectedcells, † P<0.001). Data in the graph depict the mean±standard deviation(SD) of three experiments. The histograms in FIG. 2C depict the totalpercent of cell proliferation in HCT-116 cells transfected with eitherSMAD7 sense (82%) or GED-0301 (47%) oligonucleotides from a singleexperiment. Thus, in the colorectal carcinoma cell lines HCT-116 andDLD-1 administration of a SMAD7 AS oligonucleotide resulted in asignificant decrease in cell proliferation.

Distribution of cells in different cell cycle phases was also analyzedin HCT-116 cells following transfection of GED-0301. HCT-116 cells wereeither not transfected (Untr) or transfected with SMAD7 sense orGED-0301 oligonucleotides. HCT-116 cells were transfected with SMAD7sense or GED-0301 oligonucleotides at 1 μg/ml. Twelve hourspost-transfection, cells were washed with PBS and cultured in freshmedium for an additional 24 hours. The percentages of cells in differentphases of the cell cycle was then assessed by flow cytometry. Astatistically significant increase in the percentage of cells residingin S phase, and a statistically significant concomitant decrease in thepercentage of cells constituting the G2/M population were observed incells transfected with GED-0301 compared to controls (FIG. 2D;GED-0301-transfected cells vs SMAD7 sense-transfected cells, for Sphase, *P=0.001; for G2/M phase, **P=0.01). One of three representativeexperiments in which similar results were obtained is shown. Theseresults demonstrate that knockdown of SMAD7 protein facilitated byadministration of the SMAD7 antisense oligonucleotide GED-0301 resultedin altered cell cycle phase population distribution in colorectal cancercells.

Progression through the cell cycle is regulated by cyclin dependentkinases (CDKs), which associate with activating partners (i.e., cyclins)to regulate the activity of proteins that play roles in cell cycleprogression. CDK activity is itself modulated by both inhibitory andactivating phosphorylation. In particular, it is known that theCDK-cyclin complex can be inhibited by phosphorylation of Thr-14 andTyr-15 residues within the ATP-binding pocket of the CDK. CDK2 plays acentral role in the control of S-phase, binding to either cyclin E orcyclin A to regulate the G1/S transition and S phase progression,respectively.

The phosphorylation state of CDK2 was analyzed in HCT-116 followingtransfection with GED-0301. HCT-116 cells were either left unstimulated(Uns) or transfected with SMAD7 sense or SMAD7 AS oligonucleotide.HCT-116 cells were transfected with SMAD7 sense oligonucleotide at 2μg/ml or SMAD7 AS oligonucleotide at 1 μg/ml or 2 μg/ml. Six hourpost-transfection, cells were washed with PBS and re-cultured with freshmedium for an additional 16 hours. p-CDK2 (Thr-14/Tyr-15), CDK2, andβ-actin levels were analyzed by Western blotting of cell extracts. Oneof three representative experiments in which similar results wereobtained is shown in FIG. 3. The blot in FIG. 3 demonstrates thattransfection of HCT-116 cells with GED-0301 resulted in a dramaticincrease in p-CDK2 (Thr-14/Tyr-15) levels compared to controls,suggesting a mechanism to explain the accumulation of cells in S phasefollowing GED-0301 administration.

Example 4: SMAD7 Antisense Oligonucleotide Administration CausesIncreased Cell Death in Colorectal Cancer Cells

Cell death was evaluated in HCT-116 cells following administration ofthe SMAD7 antisense oligonucleotide GED-0301 to determine whether theobserved changes in cell cycle distribution correlated with activationof cell death programs. To investigate cell death, HCT-116 cells wereeither left untreated (Untr) or transfected with SMAD7 sense or GED-0301oligonucleotide at 1 μg/ml for twelve hours. Cells were then washed withPBS, cultured for an additional six hours, re-washed with PBS, andcultured in fresh medium for another 24 (FIG. 4A, top panel) to 48 (FIG.4A, middle panel) hours. Cell death was assessed by flow cytometryanalysis of AV and/or PI staining. A significant increase in percent ofcell death as assessed by the combined AV−/PI+, AV+/PI+, and AV+/PI−populations was observed at 48 hours for HCT-116 cells transfected withGED-0301 compared to cells transfected with SMAD7 sense oligonucleotide(FIG. 4A, middle panel; SMAD7 sense vs GED-0301, P<0.001). Results areexpressed as the mean±SD of three experiments. Representative dot-plots(FIG. 4A, bottom panel) show the percentages of AV- and/or PI-positiveHCT-116 cells 48 hours post-transfection.

To further evaluate activation of cell death pathways in colorectalcancer cells following SMAD7 knockdown, the percent of cells expressingactive caspase-3 was investigated in cells transfected with GED-0301.HCT-116 cells were either left untreated (Untr) or transfected withSMAD7 Sense or GED-0301 oligonucleotides at 1 μg/ml for twelve hours.Cells were then washed with PBS and cultured for another six hours withfresh complete medium before being washed with PBS and cultured in freshmedium for an additional 16, 24, or 36 hours. Activation of caspase-3was then assessed by flow cytometry. The dot-plots in FIG. 4B show anotable increase in active caspase-3 in GED-0301-transfected cellscompared to SMAD7 sense-transfected and untransfected cells. Moreover,GED-0301 transfection resulted in progressively higher percentages ofactive caspase-3 cells at each time point. These results demonstratethat in the HCT-116 colorectal cancer cell line, administration ofGED-0301 resulted in a significant increase in the percent of cellsundergoing cell death or expressing active caspase-3 compared tocontrols.

To determine whether cell death could be blocked in cells transfectedwith GED-0301, cells were cultured in normal media or in the presence ofthe pan-caspase inhibitor Q-VD-OPH or dimethyl sulfoxide (DMSO), for onehour, and then either left untreated or transfected with SMAD7 sense orGED-0301 oligonucleotides for 36 hours. The percent of cells expressingactive capase-3 was assessed by flow cytometry. While no significantdifference was observed in any of the untransfected or SMAD7sense-transfected groups, a significant decrease in the percent of cellsexpressing active caspase-3 was observed between GED-0301-transfectedcells exposed to no drug or exposed to Q-VD-OPH (FIG. 4C; No drug vsQ-VD-OPH, P=0.002).

The same protocol was used to assess the percent of cell death, exceptcells were assessed 48 hours post-transfection and percent of cell deathwas assessed by looking at the combined AV- and/or PI-positivepopulations within the total cell population. While no significantdifference was observed in any of the untransfected or SMAD7sense-transfected groups, a significant decrease in the percent of cellsundergoing cell death was observed between GED-0301-transfected cellsexposed to no drug or exposed to Q-VD-OPH (FIG. 4D; No drug vs Q-VD-OPH,P=0.008).

To determine whether GED-0301-induced HCT-116 cell growth arrest issecondary to induction of cell death, cell proliferation was assessed inGED-0301-transfected cells exposed to Q-VD-OPH. Cells were cultured innormal media or in the presence or absence of Q-VD-OPH or DMSO for onehour, and then either left untreated or transfected with SMAD7 senseoligonucleotide or GED-0301. After 24 hours the percentage ofproliferating cells was assessed by flow cytometry. Regardless ofQ-VD-OPH exposure, all GED-0301-transfected cell populations showed adecrease in percent of proliferating cells compared to the SMAD7sense-transfected and untransfected groups, demonstrating that celldeath is a secondary effect of decreased proliferation in colorectalcancer cells subjected to SMAD7 protein knockdown (FIG. 4E). Resultsshown in FIG. 4C-4E are the mean±SD of three experiments.

Example 5: In Vivo SMAD7 Protein and mRNA Expression are Increased in aMouse Model of Colorectal Cancer

SMAD7 protein and mRNA expression were evaluated in tumoral andnon-tumoral areas of mice with colitis-associated colorectal cancer todetermine whether induction of colorectal cancer in vivo was associatedwith increased SMAD7 levels. In the model utilized herein, C57BL/6J micewere administered AOM followed by repeated DSS ingestion (AOM+DSS),causing colonic inflammation and subsequent development of multiplecolonic tumors. Animals were sacrificed 84 days after AOM+DSS treatment.SMAD7 protein levels were assessed by immunostaining of non-tumoral andtumoral areas of tissue collected from mice with colitis-associatedcolorectal cancer. FIG. 5A shows a clear increase in SMAD7immunostaining in tumoral areas of AOM+DSS-treated mice. The image istaken from one of three experiments conducted. SMAD7 mRNA expression wasalso increased in tumoral areas compared to non-tumoral areas ofAOM+DSS-treated mice as assessed by real-time PCR (FIG. 5B). Values inFIG. 5B represent the mean±SEM, and six mice were included in eachgroup. Thus, increased SMAD7 mRNA and, in particular, protein expressionin vivo was correlated with induction of colorectal cancer exclusivelyin tumoral tissue.

Example 6: SMAD7 Antisense Oligonucleotide Administration Results inDecreased Tissue Explant Cell Proliferation

To assess the effect of SMAD7 antisense oligonucleotide-facilitatedknockdown on cell proliferation in tissue, H&E staining or PCNAimmunostaining was performed on sections from ex-vivo colorectal cancertissue explants. Fresh tissue explants were transfected with SMAD7 senseor GED-0301 oligonucleotides for 36 hours and then stained. FIG. 6 showsrepresentative images of H&E- and PCNA-stained sections from freshlyobtained explants. A clear decrease in both H&E staining and PCNAimmunosignal was observed in tissue explants transfected with GED-0301compared to explants transfected with SMAD7 sense oligonucleotide,demonstrating that GED-0301 efficiently reduced cell proliferation intransfected tissue. Images from one of two representative experiments isshown.

Example 7: SMAD7 Antisense Oligonucleotide Administration ReducesColorectal Cancer Tumor Growth and Cell Proliferation In Vivo

An HCT-116 xenograft model was used to assess the effect of GED-0301administration upon colorectal cancer tumor development and colorectalcancer cell proliferation. To that end, Rag-1^(−/−) mice were inoculatedwith HCT-116 cells. One week post-inoculation, mice received a singleintraperitoneal injection of either PBS (FIG. 7A, CTR) or 100 μg ofFITC-conjugated GED-0301 (FIG. 7A, GED-0301 FITC-conjugated). Mice weresacrificed 24 hours after reagent injection, tumors were excised, andFITC-conjugated GED-0301 distribution was assessed by immunofluorescentsignal. FITC signal clearly co-localized with nuclear signal fromxenograft cells (FIG. 7A, Dapi/FITC panel).

SMAD7 protein signal was also assessed in xenografts from animalssubjected to GED-0301 administration. HCT-116 cells were inoculated intoRag-1^(−/−) mice, and animals were treated intraperitoneally with eitherSMAD7 sense or GED-0301. Both oligonucleotides were administered at 100μg/mouse every day, starting 7 days after HCT-116 injection. Mice weresacrificed 21 days after HCT-116 cell inoculation. Western blotting oftotal protein extract from xenograft tissue revealed observablydecreased levels of SMAD7 signal in samples from GED-0301-treated micebut not SMAD7 sense-treated mice (FIG. 7B). β-actin was used as loadingcontrol. Thus, GED-0301 was able to knock down levels of SMAD7 proteinin colorectal cancer xenograft cells in vivo.

Xenografts harvested in mice subjected to the same protocol describedabove were analyzed for the volume of tumors generated in each group. Asignificant decrease in tumor volume was observed in HCT-116-inoculatedmice treated with GED-0301 compared to mice treated with SMAD7 senseoligonucleotide (FIG. 7C, SMAD7 sense vs GED-0301, P<0.001). Each pointon the graph in FIG. 7C represents the volume of a single tumor.Horizontal bars indicate the median tumor volume in each group. One oftwo independent experiments in which similar results were obtained isshown. FIG. 7C provides a representative photograph of xenograftsdeveloped in SMAD7 sense-treated (top row) and GED-0301-treated (bottomrow) mice.

Immunostaining for the cell proliferation marker PCNA was also performedin xenograft tissue sections harvested from mice that underwent the sameprotocol described above. Representative images of PCNA-stainedxenograft sections are shown in FIG. 7D. A decrease in PCNA signal wasobserved in tissue sections taken from xenografts developed inGED-0301-treated mice as compared to those from SMAD7 sense-treatedmice, demonstrating that SMAD7 antisense oligonucleotide-mediatedknockdown of SMAD7 protein resulted in decreased cell proliferation incolorectal cancer cells in vivo. One of 3 representative experiments isshown.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles cited herein is incorporated by reference for all purposes.

EQUIVALENTS

The invention can be embodied in other specific forms with departingfrom the essential characteristics thereof. The foregoing embodimentstherefore are to be considered illustrative rather than limiting on theinvention described herein. The scope of the invention is indicated bythe appended claims rather than by the foregoing description, and allchanges that come within the meaning and range of equivalency of theclaims are intended to be embraced therein.

1. A method of treating colorectal cancer in a patient suffering fromcolorectal cancer, the method comprising administering to the patient aneffective amount of a SMAD7 antisense oligonucleotide comprising thenucleotide sequence of SEQ ID NO: 10 to the patient.
 2. A method ofinhibiting the growth of colorectal cancer cells, comprising inhibitingSMAD7 in the colorectal cancer cells.
 3. A method of treating colorectalcancer or inhibiting the growth of colorectal cancer cells in a patientsuffering from colorectal cancer, the method comprising administering tothe patient an effective amount of a SMAD7 antisense oligonucleotidecomprising the nucleotide sequence of SEQ ID NO:
 10. 4-7. (canceled) 8.The method claim 1, wherein the SMAD7 antisense oligonucleotide isadministered parenterally.
 9. The method of claim 1, wherein the SMAD7antisense oligonucleotide is administered orally.
 10. The method ofclaim 3, wherein said administering comprises administering apharmaceutical composition comprising the SMAD7 antisenseoligonucleotide and a pharmaceutically acceptable carrier. 11-13.(canceled)
 14. The method of claim 10, wherein the pharmaceuticalcomposition is administered parenterally.
 15. The method of claim 10,wherein the pharmaceutical composition is administered orally.
 16. Themethod of claim 15, wherein the pharmaceutical composition comprises anenteric coating comprising an ethylacrylate-methacrylic acid copolymer.17. The method of claim 1, wherein the patient is a human.
 18. Themethod of claim 1, comprising administering at least 100 μg of the SMAD7antisense oligonucleotide.
 19. The method of claim 18, comprisingadministering from 35 mg to 500 mg of the SMAD7 antisenseoligonucleotide.
 20. The method of claim 3, wherein the patient is ahuman.
 21. The method of claim 3, comprising administering at least 100μg of the SMAD7 antisense oligonucleotide.
 22. The method of claim 21,comprising administering from 35 mg to 500 mg of the SMAD7 antisenseoligonucleotide.
 23. The method of claim 1, wherein all of theinternucleoside bonds of the nucleotide sequence of SEQ ID NO: 10 arephosphorothioate bonds.
 24. The method of claim 3, wherein all of theinternucleoside bonds of the nucleotide sequence of SEQ ID NO: 10 arephosphorothioate bonds.